Cells that make up tissues and organs exist and communicate within a complex, three-dimensional (3D) environment. The spatial orientation and distribution of extracellular matrix (ECM) components directly influences the manner in which cells receive, integrate, and respond to a range of input signals.1 As such, cellular interactions with ECM molecules and/or other cells have been extensively investigated for fundamental studies in development, cell motility, differentiation, apoptosis, paracrine signaling, and applications in tissue engineering.2,3 There has been tremendous effort toward the design and fabrication of 3D scaffolds that mimic ECM properties and induce tissue formation in vitro, utilizing various biomaterials, biodegradable polymers,4 collagen,5 and hydrogels.6,7 Among the major challenges facing the use of these technologies for tissue engineering are the abilities to force contact between multiple cell types in 3D to control the spatial and temporal arrangement of cellular interactions and tailor and mold the biomaterial to recapitulate the 3D, in vivo environment under laboratory constraints. Without the use of engineered scaffolds in culture, most cells are unable to form the necessary higher-order 3D structure required for the anatomical mimicry of tissue and are limited to random migration, generating two-dimensional (2D) monolayers. As a result, several approaches, including the use of dielectrophoretic forces,8,9 laser-guided writing,10-12 surface manipulation,13 and a number of lithographic printing techniques14-17 have been integrated with 3D scaffold designs to produce multi-type cellular arrays9,11,17,18 or 3D cell clusters or spheroids.7,8,13 In a recent study, 3D aggregates consisting of multiple cell types were formed within a hydrogel matrix through DNA hybridization after cell surfaces were engineered with complementary short oligonucleotides via a metabolic labeling approach.7 However, for some applications, the presentation of cell-surface DNA may not be stable for extended time periods in cell culture or in vivo.
Cell-surface engineering methodologies have primarily been of interest in molecular biology. As such, biosynthetic approaches have been employed to introduce different functional groups on cell surfaces. In a pioneering study, an unnatural derivative of N-acetyl-mannosamine, which bears a ketone group, was converted to the corresponding sialic acid and metabolically incorporated onto cell-surface oligosaccharides, resulting in the cell surface display of ketone groups.19 However, metabolic or genetic methods may alter many of the biochemical pathways required for normal cell function and not all cell lines possess this metabolic machinery. Thus, there is a growing demand for general tools that can provide simple alternatives to the complex genetic and biosynthetic methods. Other approaches to cell-surface engineering have also been undertaken to incorporate a functional group into a target biomolecule, such as an endogenous protein, utilizing a cell's biosynthetic machinery.20,21 These strategies aim to produce a site that can then be covalently modified with its delivered counterpart or probe. However, most of these protein-based tags are large and bulky and become problematic when interacting with the other glycans and biomolecules on the cell suface.22,23 Additionally, the perturbation of cellular physiology with biomolecules at the cell surface may result in the interference of significant biochemical pathways or cellular functions.24,25.
Membrane fusion processes are ubiquitous in biology and span multi-cellular communication, extracellular signaling, the reconstruction of damaged organelles, and integration of cells into complex tissues and organs.26 As a result, there has been much interest in developing model systems to mimic biological membranes to investigate the mechanisms of fusion and for use in various biotechnological applications. For example, cells secrete and display proteins and lipids during vesicle trafficking events that either diffuse into the ECM or become components of the cell membrane after fusion.27 Naturally, lipid vesicles provide an ideal platform for such studies and have been widely used to examine various membrane-related processes, including fusion.28-39 In order for fusion to occur, the membranes must be brought into close proximity, followed by bilayer destabilization.31 Fusion of such lipid vesicles or liposomes can be initiated by using divalent cations, polycations,32 positively charged amino acids33 and membrane-disrupting peptides.34,35 Historically, synthetic chemical agents have also been employed to fuse vesicle membranes36-39 through non-specific interactions. However, recent efforts to improve selectivity and control over vesicle fusion have been achieved through the use of small, synthetic molecular recognition pairs.40-41 Since vesicle fusion is a natural process and has been shown to influence the construction of cells into multicellular organisms, much research has focused on using liposomes to deliver cargoes, reagents, nanomaterials, and therapeutic agents to cells.
Noncovalent cell-surface engineering strategies via cationic graft copolymer adsorption and a fluorescent cell labeling technique via cationic and aromatic lipid fusion have been previously reported.42 